Smartphone-Based Lab-on-a-Chip Sensor for Flu Detection

Jeong-Yeol Yoon

Influenza, commonly referred to as flu, is so common that we often forget its danger. However, according to the CDC, it is one of the top ten causes of death in the United States (www.cdc.gov/nchs/fastats/lcod.htm). Flu jumps from one species to another through coughs or sneezes. In the past couple of decades, we have witnessed two major outbreaks of lethal flu variants: bird flu (influenza A/H5N1) and H1N1 flu (influenza A/H1N1). Since their initial symptoms are very similar to those of other seasonal flu variants, early diagnosis is critical.

The current gold standard in flu detection is reverse transcription polymerase chain reaction (RT-PCR) in which a specific sequence of genetic material from flu is copied about a million times. An alternative method, called an immunoassay, takes advantage of antibodies specific to the influenza virus. Both processes require a variety of laboratory equipment and a series of pipetting and liquid handling operations, all of which take several hours. They are preferably performed in a laboratory setting, which involves an additional time lag.

Lab-on-a-chip (LOC) detects flu

For early diagnosis of influenza, it is desirable to conduct RT-PCR or an immunoassay at the point-of-care level, or even in the field. The lab-on-a-chip (LOC) is the perfect instrument for this. An LOC is essentially a network of channels and wells that is etched onto a silicon or polymer substrate in order to build a miniature laboratory. The LOC enables sample handing, mixing, dilution, separation, staining, and detection within a single, integrated system and is perfectly suitable for chemical or biological assays.

Both RT-PCR and immunoassays, including flu detection, have been demonstrated in LOCs. Final detections in an LOC can be made electrochemically or optically, but optical detection is gaining popularity due to its high sensitivity and better specificity. Back in the old days, the optical detection system incorporated into an LOC was often much bigger than the LOC itself, and it required a separate laptop computer to process and analyze the signals. These days, optical detection in an LOC can be implemented through the use of an LED light source, a pair of optical fibers, and a miniature spectrometer. One example is particle immunoagglutination assay in an LOC, combined with Mie scatter detection, which has been demonstrated by our lab. Recently, our lab fabricated optical waveguide channels to guide the on-chip light in a more reproducible manner to further improve the sensitivity down to a single-cell level or 1 picogram scale.

Smartphone-based optical detection in LOC

In fact, this kind of optical detection can now be performed with the use of a single smartphone. Modern smartphones possess both a light source and a light detector: a white LED used as a camera flash and a digital camera used as an image detector. The white LEDs in many smartphones are very bright, offering sufficient power for use as a light source for optical detection. Meanwhile, the resolution and sensitivity of smartphone digital cameras have already surpassed those of standalone, compact digital cameras.

Our laboratory has recently replaced the separate light source, optical fibers, and miniature spectrometer of an LOC system with a single smartphone for measuring Mie scatter of particle immunoagglutination assays. The light from the smartphone’s white LED is guided through a series of lenses and mirrors and finally into the optical waveguide channel of the LOC. Mie scatter from the LOC microchannel is likewise collected with an optical waveguide channel and delivered to the smartphone’s digital camera. A separate laptop computer is not necessary because the data collection and processing can be done using a software application designed for the smartphone.

It is also possible to use the same smartphone-based optical detection for the fluorescent quantification of RT-PCR, eliminating the need for a separate gel electrophoresis step. If an appropriate LOC pattern is prepared to conduct various portions of RT-PCR, then the same optical detection components can be implemented around the LOC to quantify the fluorescent signals during thermocycling (of RT-PCR). Whether it is based on RT-PCR or immunoassay, the smartphone-based LOC can provide a handheld, near-real-time, and sensitive solution for flu detection at the point of care or in the field.

Sampling for flu detection

Samples for flu detection are typically collected with a swab from the patient’s nose or throat. These samples are highly viscous and contain lots of glycoproteins and antibodies that may interfere with both RT-PCR and immunoassays.

Appropriate sample pretreatments are necessary, particularly centrifugation or filtration (plus gene extraction for RTPCR). Another sampling method is the collection of aerosols over a substantial period of time, since influenza spreads as aerosols through coughs or sneezes. Commercial air samplers that use filter paper and a vacuum pump can be used to capture these aerosols. Because these samplers have been shown to successfully capture airborne pathogens, including influenza, our laboratory has used them in conjunction with LOC biosensors. While these aerosol samples may contain much less mucus, there are large amounts of dust particles that can interfere with RT-PCR or immunoassay. Therefore, centrifugation or filtration is still necessary.

Although centrifuging has been demonstrated on an LOC platform, it is generally considered cumbersome and inappropriate at the point-of-care or field-deployable level. Filtration, however, can easily be implemented, especially with a syringe equipped with a filter (syringe filter). There are a couple of drawbacks with this approach: it requires human labor, and the person-to-person variation can lead to significant variances in assay results. LOC-based filtration could offer an alternative, eliminating human labor and ensuring reproducible assay results. However, implementing porous membrane structures within a microchannel has been a serious challenge in LOC applications, and it has not always been successful when dealing with smaller biomolecules such as viruses (including influenza).

Paper-based LOC for flu detection

LOCs are typically fabricated using silicon or plastic substrates, but they can also be fabricated on paper (cellulose fibers) as a low-cost, field-deployable, and easy-to-use alternative to conventional LOC devices. There is also another major advantage of paper-based LOCs for flu detection: the paper fibers can serve as a filter for various types of flu samples.

If a paper LOC can be used for flu detection, why can’t we use it with smartphone-based optical detection? Smartphone detection can also eliminate issues related to the inhomogeneity of cellulose fibers since it can average the optical signals over a substantial area and over different colors (when a white LED flash or white ambient light is used). This is not possible with conventional spectrophotometric analyses. Toward this end, our laboratory has recently created a multichannel paper LOC combined with smartphone detection. The paper LOC is preloaded with antibody-conjugated submicron particles, and the sample flows through the paper fibers by capillary force while unwanted contaminants are effectively filtered out. The smartphone is tilted at a predetermined, optimized angle for the best Mie scatter detection using a software application that measures the extent of Mie scatter coming from each channel. Although this work was demonstrated for Salmonella detection, it can easily be adapted to monitor flu.

News to date

Currently, there is much discussion about using smartphones for healthcare applications, commonly referred to as “mHealth.” Some of these discussions focus on smartphones’ data storage ability for medical records (especially imaging data), especially considering the “cloud” environment in conjunction with WiFi or 3G/4G LTE. Other discussions have focused on attaching a small objective lens to the smartphone camera so that the smartphone can be used as a portable microscope. More recently, a smartphone has been used as an optical sensor to read a signal from a capillary channel (for simple biological assays such as cell sorting) or from a lateral-flow assay (a common format for pregnancy tests). Combining more advanced types of LOC, especially paper-based LOCs, with smartphone detection is something entirely new. It has great potential for monitoring the presence of flu in humans, animals, and the environment, and it requires only a piece of paper and a smartphone app. And it is not limited to flu; smartphone-based detection with a paper LOC can be applied to any type of public health or environmental monitoring application.